Method of preparing a reaction mixture and related products

ABSTRACT

The invention relates to a method of preparing a reaction mixture for Polymerase Chain Reaction (PCR) assay and a solution set for PCR. The method comprises providing a sample solution comprising a biological sample to be amplified in said PCR assay and first colorant providing the solution a first color, providing a reagent solution comprising at least one other substance required for performing said assay and second colorant providing the solution a second color different from the first color, and mixing the sample solution and the first reagent solution for providing a mixed solution to be subjected to the PCR process, the mixed solution having, due to said first and second colorants, a third color different from the first and second colors. The invention significantly aids in pipetting PCR assays.

RELATED APPLICATIONS

This application claims priority to copending U.S. application Ser. No.13/499,518, which is a national stage application of PCT/FI2010/050772filed Oct. 4, 2010, which claims priority to Finnish application SerialNo. 20096013 filed Oct. 2, 2009, each of which is expressly incorporatedby reference herein in its entirety.

FIELD OF THE INVENTION

The invention relates to pipetting of (bio)chemical reagents. Inparticular, the invention relates to a method of pipetting of reagentsto microwells for Polymerase Chain Reaction (PCR) amplification, inparticular quantitative PCR (qPCR) amplification. In addition, theinvention relates to new products for aiding pipetting.

BACKGROUND OF THE INVENTION

When pipetting the PCR reaction, all necessary components, i.e.reagents, can be added to the reaction tube one by one, or preferably byfirst combining at least some of them as a master mix followed bydividing this mixture to multiple samples. One of the components thatusually must be added separately is the sample under study. The numberof samples, tubes or sample wells in a microtiter plate can be hundredsor even thousands per setup.

Adding reagents correctly, i.e. in the right order and amount, iscrucial for obtaining valid results not only in PCR but also in manyother (bio)chemical reactions. Failed experiments result in loss of timeand money. Economic importance can be huge. This is because of waste ofingredients, plastic ware and personnel working hours. Moreover, delaysin obtaining the results of the experiments may be significant. Therehave been various solutions to this well-known problem.

There are mechanical solutions to the problem. The art acknowledgesvarious automated pipetting robots, multichannel pipettes and guidancesystems (e.g. Finnzymes PIKO® Light Plate, BIOTX WELL AWARE™) used withsample tubes and plates.

For several years there have also been available PCR master mixes orbuffers that contain some visible dye to help pipetting and tracking ofelectrophoresis runs. These mixes typically also have some component toincrease density of the solution to help in electrophoresis gel loading.

U.S. Pat. No. 6,942,964 discloses a product using a pipetting aid dyewhich is also used as a gel loading and tracking dye. The colorant hasbeen incorporated with the polymerase, which helps the user to seewhether the polymerase has been pipetted to the PCR mix or master mix. Asimilar product is BioLine Accuzyme Red.

USB Corporation's RubyTaq features a polymerase including a mixture oftwo dyes which are separated during the agarose gel run: magenta (runsbetween 500 bp [2% gels] and 1500 bp [0.8% gels]) and yellow (runs lessthan 10 bp).

Fermentas and Promega have also added a colorant to the enzyme reactionbuffer. Fermentas' DREAMTAQ™ Green reaction buffer can be seen as green,but the color separates into a blue and yellow bands during gelelectrophoresis. Promega GOTAQ® and GOTAQ® Green Mastermix behavesimilarly.

There are also products available where the dye added to the polymeraseis not intended to help in the electrophoresis phase. Examples includeABgene RED HOT®.

NEB provides a product (CRIMSON TAQ®) featuring a dye Acid red added tothe DNA polymerase reaction buffer. The product also uses 6% dextran asa density enhancer.

Qiagen's CORALLOAD® dye is available both as a concentrate in a separatetube for being added to an uncolored master mix, and also as an optionalready-made 10×PCR buffer. It contains two gel tracking dyes (orange andred).

KR 2002/0045167 discloses freeze-dried PCR mixes containing a colorantto confirm dissolution of the PCR components. U.S. Pat. No. 6,153,412discloses also freeze-died reaction mixtures which are used foridentifying the existence of a lyophilized PCR reagent and to ensurecomplete mixing of the PCR reagent and the test sample. U.S. Pat. No.5,565,339, on the other hand, discloses the use of a dye in a hot startwax, which does not dissolve into the reaction mixture.

ABSOLUTE™ Blue QPCR Master Mix contains an inert blue dye to easepipetting in reaction set-up.

Also WO 2007/088506 discloses a dyed master mix.

Applied Biosystems has ROX passive reference dye included in their qPCRproducts. The purpose of the dye is to provide a steady fluorescencelevel which can be used to normalize against any non PCR relatedfluorescence variation between the different reactions and in one sampleduring a reaction. The method is also suggested to normalize at leastpartly against deviations in pipetting accuracy.

All but the three last ones of the products mentioned above aresuggested to be used only in traditional end point PCR. In addition tothe colorants, they typically contain a density enhancer to get thesample material into the bottom of the gel wells (see e.g. U.S. Pat. No.6,942,964). Without the density enhancer, samples would disperse intothe surrounding liquid.

Colorants used in end-point PCR are generally not compatible withquantitative PCR (qPCR). This is usually because they prevent real-timeoptical measurements of the ongoing reaction. In particular, the dyestypically have a spectrum which overlaps with the detection wavelengthsof qPCR fluorescence or their absorbance is too high. Generalrequirements for the dyes used include non-inhibitory effect on the PCRreaction or stability in the reaction pH.

An additional disadvantage of the abovementioned solutions in which thedye is provided in the master mix or in the polymerase is that they arenot able to provide help for pipetting the samples (i.e. in PCR thematerial to be amplified). Sample pipetting is, however, the step wherekeeping track of the process is most important, and most difficult.

The various mechanical systems available cannot solve the problementirely. They are expensive and pipetting errors cannot always be seenvisually mainly because of volume differences, which means the errormight not be detected until after obtaining failed results.

Thus, there is a need for enhanced pipetting aids. In particular, thereis a need for such pipetting aids which can also be applied for thesample pipetting phase.

SUMMARY OF THE INVENTION

It is an aim of the invention to provide a novel solution for aidingpipetting during preparation of PCR assays and in particular for makingthe detection of errors in various stages of pipetting easier.

The aim of the invention is achieved by the invention as defined in theindependent claims.

In the pipetting stage of PCR assays, at least two reagent solutions aremixed for obtaining the final mixture which is subjected to PCR. Theinvention is based on the idea of coloring the reagent solutions withdifferent initial colorants, which, upon mixing produce adistinguishable color different from the colors of the initialcolorants.

Thus, one can tell directly by the color of the solution, whether it isthe first reagent solution, the second reagent solution or the mixtureof these.

More specifically, the method comprises

-   -   providing a first reagent solution comprising at least one        substance required for performing the assay and a first colorant        providing the solution a first color,    -   providing a second reagent solution comprising at least one        other substance required for performing said assay and a second        colorant providing the solution a second color different from        the first color,    -   mixing the first and second reagent solutions for providing a        mixed solution to be subjected to the PCR process, the mixed        solution having, due to said first and second colorants, a third        color different from the first and second colors.

In a typical application, one of the reagent solutions is a samplesolution, that is, a solution containing or intended to receive abiological sample to be amplified in the PCR assay, and the other of thereagent solutions contains some other at least one other substancerequired for performing the assay, for example the polymerase solutionor master mix. The sample solution may be a buffered solution(hereinafter “a sample buffer solution”). Thus, a microwell having afirst color indicates that there is only sample solution without otherreagents, e.g. master mix, in the well. A microwell with second colorindicates that master mix has been added but there is no sample yet.Finally, a microwell with third color implies that sample has beenproperly added to the master mix. The inspection of the color can bemade visually or by automatic optical means.

In one embodiment, one of the reagent solutions is an elution buffer,such an elution buffer used in combination with a nucleic acidpurification kit.

In one embodiment, one of the reagent solutions is a dilution bufferused to facilitate lysis of a solid-state sample to release nucleicacids. The reagent solution can also be used to dilute, digest orprecipitate released components before PCR. Thus, it is possible to usethe invention when pipetting direct PCR assays.

In further embodiments, one of the reagent solutions is a solution usedin cDNA synthesis reaction, reverse transcriptase reaction or bisulphitereaction.

In one embodiment, one of the reagent solutions is some other solutionused for preparing the sample for the PCR process.

The invention also provides a new use of dyes for producing two or morecolored PCR reagent solutions, which are capable of forming a mixedsolution having a color distinguishable from the initial colors of thereagent solutions.

Further embodiments are the subject of the dependent claims.

A particular aim of the invention is to achieve a pipetting aid solutionwhich is compatible with quantitative PCR. This is achieved by usingsuch colorants and colorant concentrations which do not significantlydisturb the fluorescent processes, i.e. excitation and emission, oroptical detection used in qPCR. In particular, the reaction mixturesubjected to qPCR is transparent or translucent at least at the qPCRexcitation and emission wavelengths. This generally means that themaximum absorbance of the of the reaction mixture is less than 0.5, inparticular less than 0.15 (measured using 1 mm light path) and that theabsorption window of the colorant does not overlap, at leastsignificantly, with the excitation or emission wavelengths of thefluorescent qPCR dye(s) or modified DNA oligonucleotide probe(s) used.

In one embodiment, a reaction mixture for quantitative PCR is prepared,the reaction mixture comprising fluorescent dye, primer or probe, andwherein the absorbance peaks of any of said colorants do not overlapwith the emission or excitation wavelength of said fluorescent dye,primer or probe. If overlap exists, it should not significantly weakenthe qPCR signal, generally implying that the total absorbance of thereaction mixture at said wavelengths is less than 0.05, preferably lessthan 0.03, in particular less than 0.1.

The invention provides considerable advantages. As the initial solutionsand the resultant solution are mutually of different colors, one notonly distinguish between the initial solutions, but also between theinitial solutions and their mixture.

In addition, from colored solutions one can be quickly perceive whetherthe solutions have been properly mixed and whether there are significantdeviations from the desired reaction volume.

Moreover, the colors make it easier to see if there is any liquidsplashed or spilled in wrong places where they could potentially causecontamination, microwell sealing problems etc. Especially with adhesivesealing films applied on microtiter plates before thermal cycling, anyliquid in the sealing contact can compromise the seal and thus the wholePCR assay.

The present invention can also be used together with the mechanicalsolutions to lower their error rate even more, if the pipetting stepsperformed are visualized using the colorants. When using pipettingrobots, it is possible to add a quality check step based on opticaldetection after desired steps, or the volume and color of the reagentscan be quickly checked visually.

For the above reasons, dyes and other colorants used in the presentmanner can help keeping track in reaction setup and especially duringloading reagents into reaction plate. Thus, the approach providesconsiderable help and increased certainty for pipetting samples.

In qPCR there is no need to load the amplified products into a gel aftera PCR reaction. Thus, a density enhancer is not needed. Consequently,the present solutions may be free of density enhancer or contain onlyminor amounts of density enhancer (i.e. less than required for gelelectrophoresis).

According to one embodiment, there is provided, in addition to theabovementioned first and second reagent solutions, one or moreadditional reagent solutions comprising additional colorants providingthe solutions different colors. The solutions are capable of forming, onmixing, additional solutions having, due to said additional colorants,further distinguishable colors. Thus, the invention can be used not onlyfor aiding the pipetting in one particular stage of the process, e.g.pipetting of the of the sample to master mix, but also for aiding thepipetting during other steps, in particular the steps previous orsubsequent to the sample pipetting step.

In more detail, the method may comprise providing a third reagentsolution comprising at least one further substance required forperforming said assay, the third reagent solution containing a thirdcolorant providing the solution a fourth color different from the first,second and third colors mentioned above, and mixing the third reagentsolution with the first and second reagent solutions for providing amixed reagent solution having, due to said first, second and thirdcolorants, a fifth color different from the first, second, third andfourth colors. In particular, the first reagent solution may be asample, the second reaction solution the master mix and the thirdreagent solution may be a primer solution. The order of application isnot essential, unless otherwise defined in assay instructions.

Alternatively, to the above the method may comprise providing a thirdreagent solution containing third colorant providing the solution afourth color different from the first, second and third colors, andindividually mixing the first reagent solution with said second andthird reagent solutions for obtaining first and second mixed solutionshaving third and fifth colors, respectively, different from each otherand the first, second and fourth colors. For example, the second reagentsolution may contain one set of primers and the third reagent solutionmay contain a second set of primers. In this embodiment too, the colorsof all initial ingredients and all resultant mixtures are unique.

The two abovementioned embodiments can also be chained such that thesecond and third reagent solutions are ultimately individually mixedwith a reagent solution which is itself prepared by mixing at least twodifferent colored reagent solutions. Other kinds of combinations arepossible too.

A “reagent solution” is any solution containing at least one reagentneeded or advantageously used for PCR purposes. Most typical ingredientsare polymerase, nucleotide, primer, ions, magnesium, other salt, pHbuffering agent, dNTPs or fluorescent qPCR dye or probe,oligonucleotide, nucleic acid binding agent, a nucleic acid template.The reagent may also be other polymerase reaction additive, which has aninfluence on the polymerase reaction or its monitoring.

The term “sample solution” covers both buffered and non-buffered samplesolutions which are still free of template or into which the template tobe amplified using PCR has already been added, unless otherwisespecified. The term “sample solution” is covered by the term “reagentsolution”.

The term “master mix” refers to a mixture of all or most of theingredients or factors necessary for PCR to occur, typically all exceptfor the template and primers which are sample and amplicon specific.Commercially available master mixes are usually concentrated solutions.A master mix may contain all the reagents common to multiple samples,but it may also be constructed for one sample only. Using master mixeshelps to reduce pipetting errors and variations between samples due todifferences between pipetted volumes. It also minimizes the time spentfor pipetting.

A qPCR master mix is a master mix intended for performing a qPCRreaction. Thus, it may contain fluorescent dye or fluorescently taggedoligonucleotide primers or probes.

The term “premix” refers to a master mix that contains all the necessarycomponents for a PCR reaction except for the template.

The term “color” herein means any detectable spectral response (of asolution) to white light in the visual range. Thus, there is at leastone wavelength range in the absorbance spectrum of the solution whichprovides a colored visual appearance for the solution (in contrast tothe nearly 100% transmittance of water). White, black, and shades ofgray are herein counted as colors. As will be shown later, an absorbancehigher than about 0.01 (1 mm light path) gives a visually perceivablecolor for a solution whereas an absorbance higher than about 0.001 (1 mmlight path) can be relatively easily detected by hardware-based spectraldetection means.

The term “different colors” means that the colors are distinguishable,preferably by the naked eye, but at least with spectral detection means.In particular, “different colors” may have maximum peaks in theirabsorption spectrum separated by at least 30 nm. Preferably, thedifferent colors are selected from the groups of: red, yellow, blue orcyan, magenta, yellow and visually distinguishable combinations andshades thereof, such as green, orange, and violet.

The term “colorant” means any substance which is capable of beinghomogeneously mixed or dissolved within a solution and capable of givingthe solution a perceivable color. According to one embodiment, thecolorant is a dye, in particular an aqueous dye, preferably anon-oxidizing aqueous dye.

The terms “transparent” or “translucent” colorant-containing solutionrefers, in particular, to a solution which has an optical transmissionwindow at at least some fluorescence excitation and/or emissionwavelengths that can be used for performing qPCR, the wavelengthsdepending on the fluorophores, fluorescent dye(s), and/or modified DNAoligonucleotide probe(s) contained in the reaction mixture. Typically,the excitation wavelength is between 350 and 690 nm, in particularbetween 490 and 650 nm. The emission wavelength is typically between 350nm-730 nm, in particular 515 nm-680 nm. A transparent solution isoptically essentially non-diffusive, whereas a translucent solutionpasses light diffusely.

The term “sample” refers to a solid material or a solution that containsthe nucleic acid of interest or is to be analyzed for the presence ofnucleic acid of interest.

The term “dilution buffer” refers to a solution that can be used forsample pretreatment before PCR setup. Pretreatment can include samplelysis for releasing nucleic acids, dilution, binding, chemical lysis,precipitation and enzymatic digestion of some components.

The term “preparative process” refers to any reaction, pipetting step orpretreatment which yields a product which can in total or in part beused as a sample in a subsequent PCR reaction.

Typically the third color, achieved by mixing the solutions with thefirst and second colorants, is a chromatic combination of the first andsecond colors of the solutions. Thus, the third color may be produced asa sum spectrum of the spectra of the first and second colors. However,it is not excluded that the third color is formed through a more complexprocess, e.g. reaction of the first and second colorants, or due to afluorescent process, e.g. fluorescence resonance energy transfer (FRET),provided that the fluorescence wavelengths differ from those of qPCRfluorophores used.

Next, embodiments and advantages of the invention are described in moredetail with reference to the attached drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows in cross-sectional view three microwells containingcolored sample buffer, colored reagent solution and their coloredmixture, respectively.

FIG. 1B shows a top view of a microtiter plate containing empty wellsand wells containing colored sample buffer, colored reagent solution,their colored mixture and wells with optically clear liquid.

FIGS. 2a and 2b illustrate as a flow chart exemplary ways of carryingout the invention.

FIG. 3 shows a flow chart of the present process according to oneembodiment of the invention.

FIGS. 4a-4d show standard series obtained with master mix and samplewith (4 a and 4 b) and without (4 c and 4 d) pipetting aid dyes.

FIGS. 5a-5d show absorbance spectra relating to a absorbance measurementexample.

FIGS. 6a-6c and 7a-7c illustrate the use of colorant in a cDNA synthesisreaction.

DETAILED DESCRIPTION OF EMBODIMENTS

To make plate setup easier, the invention provides, according to oneembodiment, a dye combination that helps keeping track of pipettingmaster mix, samples and mixing of these, in the pipetting phase of(q)PCR process. Thus, the dyes are preferably optimized so that theywill have minimal effect on qPCR reaction (e.g. will not influence thesample or DNA polymerase used) and will not significantly affect opticaldetection of fluorescence. In other words, the dyes used are compatiblewith the qPCR assay.

Typical fluorophores used for qPCR purposes include Alexa 350, FAM™,TET™, VIC™, JOE™, HEX™, CY® 3, TAMRA™, ROX™, TEXAS RED®, CY® 5, CY® 5.5and QUASAR® 705, the emission and excitation wavelengths of which areshown in Table 1.

TABLE 1 Dye Ex Em Alexa 350 350 440 FAM 494 518 JOE 520 548 VIC 538 554HEX 535 556 Cy3 552 570 TAMRA 565 580 Cy3.5 581 596 Texas Red 583 603ROX 585 605 Cy5 643 667 Cy5.5 675 694 Quasar705 690 705

FIG. 1A illustrates the basic principle of the invention. The microwell12A contains colored sample buffer 14A having a first color (horizontallines). The microwell 12B contains colored master mix 14B having asecond color (vertical lines). The microwell 12C contains coloredmixture of the sample buffer and colored master mix, having a thirdcolor (horizontal and vertical lines) resulting from the first andsecond colors.

FIG. 1B illustrates a microtiter plate 10 which, in addition to thesolutions shown and marked as in FIG. 1A, contains empty wells 12 andnon-colored liquid 14D having no color (dots). In addition, there isshown a diluted reaction mixture 14E, which is achieved by diluting theinitial reaction mixture 14C with the non-colored liquid 14D, thediluted reaction mixture having the same basic color as the initialreaction mixture 14C, but with increased transparency, i.e. reducedabsorbance (sparse horizontal and vertical lines). As will be describedlater in more detail, not only the color, but also the degree ofdilution can be monitored according to one embodiment of the invention.

The dyes can preferably be both detected and distinguished from eachother visually, i.e. by naked eye. Thus, the different colors arespectrally relatively densely distributed and no special equipment isneeded. However, in automatic devices utilizing optical spectraldetectors or computer vision, also colors more finely distributed on thespectral scale can be used, without compromising the ability todistinguish between different colors.

According to one embodiment, the combination comprises a blue master mixand yellow sample buffer. When mixed together these form clearly greensolution. Blue color in the plate indicates that master mix has beenadded but there is no sample yet. When sample is added color turnsgreen. If solution in the well is yellow it means that there is onlysample without mastermix. According to one embodiment, the blue dyecomprises Xylene cyanol. According to one embodiment, the yellow dyecomprises Quinoline yellow. These dyes have been found to be compatiblewith the polymerase and sample buffer, respectively.

Other potential dyes comprise Brilliant Blue, Patent Blue,Indigocarmine, Acid Red 1, m-Cresol Purple, Cresol Red, Neutral Red,Bromocresol Green, Acid Violet 5, Bromo phenol blue, and Orange G. Otherpotential dyes are listed in U.S. Pat. No. 6,942,964.

According to one embodiment, the dyes are strong enough to give avisually perceivable color for the respective solutions, but weak enoughnot to disturb fluorescence detection and/or weak enough not tointerfere with gel electrophoresis migration tracking with other dyescommonly used for that purpose. For example, the abovementioned Xylenecyanol and Quinolene yellow belong to this group of dyes. Thus, if thecolored amplified reaction mixture is subjected to end-point gelelectrophoresis analysis, the colorants do not have an influence on theanalysis. Instead of that, a conventional loading buffer withelectrophoresis dye can be added to the amplified mixture. Moderatedyeing also maintains the general visual appearance of the solutionstransparent or translucent.

A suitable concentration of the dye depends on the dye itself. Accordingto one embodiment, directed to machine-aided color detection, theconcentration of the dye in the initial solution is adjusted to resultin an absorbance of 0.001-0.5 at its maximum absorption wavelength (1 mmlight path). According to an embodiment directed to visual colordetection, the concentration of the dye is adjusted to result in anabsorbance of 0.01-0.5, in particular 0.03-0.5, at its maximumabsorption wavelength (1 mm light path). According to a most preferredembodiment, the absorbance is 0.03-0.15, which ensures both visualdetectability of the color and negligible or small effect on the qPCRmeasurement even if the absorbance peak would slightly overlap with theqPCR excitation and/or emission wavelengths. It is preferred, if suchoverlap exists, that the total absorbance at the qPCR excitation and/oremission wavelength is less than 0.05, preferably less than 0.03, inparticular less than 0.01 (1 mm light path), regardless of the maximumabsorbance. As the two (or more) initial solutions have differentlycolored dyes, there is no significant cumulative absorbance at anyparticular wavelength. It should also be noted that the aboveabsorbances are the preferred absorbance of solutions diluted to thedesired PCR processing concentration. If the solutions are delivered asconcentrates, the preferred absorbances are respectively higher.

According to an alternative embodiment, at least one of the solutions isprovided with dye, which is both suitable to be used in qPCR (i.e. doesnot affect the fluorescence detection at the wavelengths used) andstrong enough to detected in gel electrophoresis, and runs on anappropriate distance at the gel with respect to the samples. Thus, aseparate loading buffer is not necessarily needed.

The sample buffer containing a dye may be delivered either as a diluteor concentration, depending on the intended use.

FIG. 2 illustrates the general concept of pipetting a colored samplesolution (step 20) and at least one reagent solution (step 21,optionally 22) into a single container and mixing the solutions (step23). The color of the mixed solution is checked (step 24) beforesubjecting the mixture to PCR (step 25). It should be noted that theremay be other pipetting and processing steps which are not shown in FIG.2 for simplicity.

Several embodiments taking advantage of the general idea of theinvention are explained below.

According to one embodiment, there are provided a plurality of coloredsample buffer solutions, in which different colorants are used to givethe sample buffers different colors. According to a further embodiment,mixing the plurality of colored sample buffers with the same coloredreagent mixture yields reaction mixtures of different colors. Thus, in amulti-sample PCR assay, one can distinguish between different samplesbased on the color of the solution. For example, a yellow sample bufferand a red sample buffer mixed with a blue master mix could give greenand magenta reaction mixtures, from which one can immediately verify notonly the proper mixing, but also the type of the sample.

According to one embodiment, there are provided a master mix and aplurality of colored primer mixes, in which different colorants are usedto give the mixes different colors (master mix: color 1, primer mixes:color 2 and color 3). Combining the primer mixes with the mister mixyields still different colored mixes (colors 4 and 6). Further, byadding a colored sample (color 7) to the mixes obtained, distinguishablePCR solutions are obtained (colors 8 and 9). In each case of theprocess, the color of the solutions is indicative of the contents of thesolution.

As illustrated in FIG. 3, according to one embodiment, there areprovided a plurality of master mixes or other premixes (steps 31, 32)which are provided with different colorants to render the premixesdifferently colored (say, premix 1: color 2, premix 2: color 3, . . .premix n: color n+1) and a sample having a further color (color 1) (step30). The premixes are individually mixed with the sample (steps 33 a, 33b) and the colors of the resulting solutions are checked (steps 34 a, 34b). The colors are preferably chosen so that each combination of apremix solution and sample solution solutions yields a unique colorrendering the solutions distinguishable from each other and the initialpremix and sample solutions. After checking (34 a, 34 b) the solutionsare, in principle, ready for PCR. It should be noted that there may beother pipetting and processing steps which are not shown in FIG. 3 forsimplicity.

More generally, there may be provided a plurality of initial solutions(each containing a component needed in the PCR reaction, e.g.polymerase, primer, ions, dNTPs or fluorescent qPCR dye or probe, orother additives) which are provided with different colorants to renderthe solutions differently colored (say, solution 1: color 2, solution 2:color 3, . . . solution n: color n+1) and a sample having a furthercolor (color 1). The colors are preferably chosen so that eachcombination of solutions yields a unique color rendering the solutionsdistinguishable from the other solutions.

Selected variations of the invention having high utility value aredescribed below.

Use of Dye in Elution Buffer

Nucleic acids for molecular biology experiments are usually purifiedfrom complex sample material. There are various methods for purificationincluding methods based on extraction, precipitation, hybridization anddifferent modes of chromatography or filtering etc. In most of thetechniques nucleic acids are either dissolved or eluted in selectedsolution. Precipitated nucleic acids can be dissolved in a variety ofsolutions. When using other method that are based on other DNAinteractions there are more requirements for the elution buffer such assuitable ionic strength. Purification methods based on DNA binding tosilica in high ionic strength conditions are widely used. Bound DNA iseluted from silica matrix with low ionic strength buffer or with purewater. Many kits based on the silica binding method are available andusually the kit contains the elution buffer. To reduce the number ofpipetting steps in the experiment workflow, the colorant can be includedin the elution buffer or the buffer provided with the kit can bereplaced with the colored buffer. By doing this, the user does not haveto add the color in a separate step.

For example, the sample buffer containing the dye can be used as asample elution buffer in combination with many commercial or homebrewDNA purification kits. The elution buffer provided with many of theavailable kits can be just simply replaced with the sample buffercontaining the dye. Alternatively, a small amount of dye concentrate canbe added in the elution buffer provided with the kit without dilutingthe sample too much.

The other colored reagent solution may be any other solution needed forthe process, as discussed above.

Use of Dye in Dilution Buffer (Direct PCR)

New enzyme technology has made it possible to significantly simplifysample preparation for PCR and it is even possible to put the unpurifiedsample directly to the PCR. However in many experiments the sample needsto be separated in to several reactions and it is often preferable to beable to store some of the sample for possible repeats or other purposes.Thus direct PCR protocols where sample is lysed and dissolved in aspecial sample buffer are very popular. In these so called dilutionprotocols the dilution buffer may contain different agents that lyse thesample. In addition to these agents a colorant can be added to thedilution buffer to make subsequent pipetting steps easier.

The other colored reagent solution may be any other solution needed forthe process, as discussed above.

To demonstrate this embodiment, two set of extractions from bovine milksamples were done with a kit based on DNA binding to silica. Onefollowing the guidelines and the other set where the elution buffer wasreplaced with 1× sample buffer with yellow dye. Purified samples wereused in qPCR and qPCR results of the described two sets were compared.No significant difference was observed.

Use of Dye in Reverse Transcription Reaction

Majority of real time PCR is done for gene expression studies. In theseexperiments the nucleic acid of interest is RNA and thus not directlysuitable template for normal qPCR. Before qPCR the RNA sample must bereverse transcribed before the qPCR step. Reverse transcription and qPCRreaction can be combined and performed subsequently in same reactionmixture. However usually the condition is a compromise and not optimalfor either of the two reactions. In most cases it is more optimal to doseparate reverse transcription reaction and use the created cDNA as atemplate in a separate qPCR reaction. In reverse transcription reactionsetup there are the same challenges of keeping track of samples duringpipetting as described for qPCR. An embodiment of the inventiondescribes how colorant can be used in cDNA synthesis reaction toovercome this challenge.

The use of colorant in cDNA synthesis reaction has also beendemonstrated as follows:

Two cDNA synthesis reaction series were prepared one with the yellowcolorant, in final concentration of 10 fold compared to theconcentration instructed for the qPCR, the other without the additionaldye. HeLa total RNA dilution series with 1000 ng, 500 ng, 10 ng 1 ng,100 pg and 10 pg dilutions were used as template. Reactions wereotherwise done according to the manual (Product number F-470,Finnzymes). A 1.5 μl aliquot of each reaction was then used as atemplate in qPCR with DyNAmo SYBR Flash qPCR master mix.

With reference to FIGS. 6a-6c and 7a-7c , two standard curves werecreated, the first (FIG. 6c ) representing the series with the added dyeand the other (FIG. 7c ) without the dye. The results show that cDNAsynthesis can be performed in presence of colorant and the quantitativenature of the reaction is maintained.

In practice, the dye can be brought into the reaction with thetranscriptase, with the sample, with the buffer solution_or separatelyas a concentrate.

Use of Dye in Bisulphite Reactions

Similarly to what is discussed above, dye can also be added to anycomponent taking part in a bisulphite treatment prior to mixing thesample thereby obtained with a second reaction solution.

As can be seen from the above examples, the dye can be present not onlywhen mixing the final PCR reaction mixture but also in preparativeprocess steps, in particular those relating to sample preparation, suchas reverse transcription reaction (e.g. in cDNA synthesis), bisulfitereaction, sample elution or sample dilution. These examples are notlimiting and, as understood by a person skilled in the art, the dye canbe introduced also into these reactions in various ways, for example,with the enzyme, with reaction buffer, with the sample or separately.

As there is color present also in the preparative process steps,pipetting of these steps is facilitated too. However, according to apreferred embodiment, at least one colored solution is brought whenmixing the final reaction solution, e.g. with the polymerase or mastermix.

FIG. 2b illustrates, at a general level, the principle of introducing atleast one colored reagent solution (step 20′) to the process prior to atleast one preparative process step 29′. The pretreatment may involve theintroduction of one or more other substances too (step 28′). Afterpretreatment, the process can be continued similarly to as explainedabove, by mixing the product (or aliquot thereof) of the preparativeprocess step with second colored reagent solution (steps 21′ and 23′),checking the color of the mixed solution (step 24′) and proceeding to(q)PCR (step 25′).

Monitoring of the pipetting process is described below.

Preferably, the different colors are distinguishable by the naked eye.However, hardware-aided optical measurements capable of distinguishingbetween colors can be utilized too.

According to one embodiment, the condition of one or more microwells tobe pipetted is checked at least once during the pipetting processautomatically by optical means capable of spectral resolution.

According to one embodiment, the condition of the microwells isautomatically checked at least two times in different stages of thepipetting process. Preferably such checking is carried out after everyseparate pipetting step.

The concentration of the colorant decreases and the color of a coloredsolution become weaker due to every addition of non-colored, i.e.optically clear, liquids. On the other hand, addition of coloredsubstances changes the shade of the color. Thus, the strength and/orshade of color of a solution within a well is indicative of the stage ofpipetting. By automatic measurement of the spectral response of themicrowell(s), the progress of the pipetting process can be monitored.

According to one embodiment, the abovementioned monitoring is carriedout using a computer program, which is adapted to compare the measuredspectral responses after the desired pipetting steps with predefinedlimits for these steps. Such limits are designed to reflect the correctshade and/or strength of the color of the solution, taking into accountthe reagents added. A microwell, for which the measured value is notwithin an accepted range, is flagged incorrectly pipetted.

The detection of the color of a solution is preferably based onabsorbance measurement. The detection instrumentation, which may be anin integral part of an automatic pipetting apparatus or a (q)PCR thermalcycling apparatus, for example, contains absorbance measurement means,i.e. a light source, a light detector and means for determining theabsorbance of the contents of a microwell at least at one wavelength orwavelength range. Preferably, the absorbance measurement means comprisea spectrophotometer. By means of the invention, the reliability ofpipetting and PCR assay can be improved, as even small changes in shadesand strengths of colors, and thus in the contents of the wells can bedetected.

According to an alternative embodiment, the detection instrumentation iscontained in a separate apparatus to which the reaction solutions can betransferred either automatically or manually after critical pipettingsteps. In the separate apparatus, a quick plate read is carried outbefore the plate is transferred for further processing.

Thus, the invention also provides an apparatus for monitoring pipetting,comprising means for receiving a microtiter plate containing a pluralityof microwells and means for measuring the optical absorbance of contentsof the microwells. The means for detecting the absorbance are adapted todetect the spectral absorbance profile of the sample (for colordetection) and/or color intensity of the sample (for dilutiondetection). Preferably, the apparatus is capable of both theabovementioned functions for being able to monitor the entire pipettingprocess. The optical detection means are preferably connected to acomputing unit which analyses and stores the measured absorbances andperforms a calculation or comparison of the measurement data withpre-stored data.

The detection instrumentation may contain a microplate-receiving blockwhich can be cooled for keeping the temperature of the reactionssolutions low enough. For most hot-start polymerases cooling is notnecessary.

Automatic detection is of particular assistance when the volume of thereaction vessel is small, that is, less than 5 μl, in particular lessthan 1 μl, as reliable visual observation of both the color and volumeof the solutions is more difficult in these cases.

The microwells may be separate or be contained in microtiter strips orplates of any known type. Preferably, the wells are manufactured fromtransparent material, allowing the visual inspection or spectralmeasurements to be carried out through the wall of the well.

Dyeing Example

Xylene cyanol as a colorant was added to DyNAmo Flash SYBR® green qPCRand DyNAmo Flash Probe qPCR master mixes from (Finnzymes, Finland) inthe concentration of 0.0026% (w/v). The result was a clearly bluetransparent solution. Quinolene yellow was added to a sample buffer inthe concentration of 0.00174% (w/v). The sample buffer contained 1 mMTris-HCl pH 8.5 and 0.1 mM EDTA. As a result, a clearly yellowtransparent solution was obtained.

The colored sample buffer and the colored master mix were mixed,resulting in a clearly green transparent mixture.

Amplification Example

FIGS. 4a-4d show standard series obtained with master mix and samplewith (FIGS. 4a and 4b ) and without (4 c and 4 d) the pipetting aid dyesof Example 1. Both series were done by amplifying human genomic DNAsequence with DyNAmo Flash Probe master mix according to the protocol inthe product manual. Primer sequences were ACCTCCAAACTGGCTGTAAC (SEQ IDNO: 1) and ATCTCCTCCTCATTGCAAAG (SEQ ID NO: 2). Detection was based onhydrolysis probe with a sequence TGGCCCCTTGAAGACAGCAG (SEQ ID NO: 3).Amplicon size was 121 bp.

From the mutual similarity of the amplification curves (FIGS. 4a and 4c) and standard curves (FIGS. 4b and 4d ) can be seen that presence ofthe dye does not affect the reaction efficiency or significantly affectfluorescence intensities.

Pipetting Example:

The present invention was utilized to implement the following pipettingsequence:

-   -   A colored (blue) 2× mix was thoroughly mixed with primers and        probes, additives and water for obtaining a premix for several        reactions.    -   The premix was pipetted to several wells of a microtiter plate        (15 μl/well).    -   Colored (yellow) DNA sample solutions were pipetted onto the        premixes (5 μl/well).    -   The color of the resulting solution was manually checked to be        correct (green).

After the above steps, the resulting solution is ready to be subjectedto (q)PCR. For qPCR, the reagents are preferably centrifuged to thebottoms of the wells.

Absorbance Measurement Examples

Absorbance of different dilutions of the dyes used in the examples aboveand dilutions of existing colored master mixes were measured andcompared to visual observation to assess the visually perceivable rangein different wavelengths. The purpose was in particular to determinevisually perceivable absorbance range with different dyes and also checkif commercially available dyes would be suitable to be used with FAM andSYBR fluorescent dyes which are probably the most popular dyes used inqPCR.

Measurements were performed with Nanodrop ND-1000 spectrophotometer,which uses 1 mm and 0.1 mm light paths.

The results, including visual detectability of color, absorbance maximaand absorbances of the samples as well as types of the solutions anddyes used in the experiments are shown in Tables 1-7 below. Tables 1-3show the results obtained with preferred dyes to be used with FAM orSYBR, whereas Tables 4-7 show comparative examples obtained withcommercially available colored PCR solutions.

In the Tables, the following denotations are used:

+++ strong color

++ color easy to see

+ weak color but visible in normal laboratory environment by naked eye

− color not visible in normal laboratory environment by naked eye

For cases denoted with asterisk (*), the absorbance peak was notcompletely well-defined or clear.

TABLE 1 Absorbance Visual maximum Product Dilution color nm (λ_(max))A_((1 mm)) A_((1 cm)) Reagent color 500x  +++ 615 Finnzymes 50x  +++ 6151.63 5x ++ 615 0.179 Xylene cyanol 1x ++ 615 0.037 0.302 (XC)   0.5x +615   0.2x − 615 0.013   0.04x 615

TABLE 2 Visual nm Product Dilution color (λ_(max)) A_((1 mm)) A_((1 cm))Sample buffer 500x  +++ 413 Finnzymes 50x  +++ 413 5x ++ 413 0.578Quinolene yellow 1x ++ 413 0.124 1.163 (QY)   0.2x + 413 0.026* 0.186  0.04x − 413 0.022*   0.02x 413

TABLE 3 Visual Product Dilution color nm (λ_(max)) A_((1 mm)) A_((1 cm))Colored reaction mix 1x ++ 413 0.128 Finnzymes 0.2x + 413 0.032 0.1860.1x + 413 0.015 0.059 G7 0.04x − 413

The absorbance maxima of the solutions of Tables 1-3 are relatively farfrom the fluorescent wavelengths of FAM and SYBR dyes. The absorbancespectrum (1× dilution) of the reaction mixture of Table 3 is shown inFIG. 5a . From the data is can be concluded, that the dyes tested provedto be suitable to be used in the initial solutions and also together ina qPCR reaction mixture as colorants with these fluorescent dyes.

TABLE 4 Visual Product Dilution color nm (λ_(max)) A_((1 mm)) A_((1 cm))Crimson Taq buffer 5x +++ 510 1.804 NEB 1x ++ 510 0.406 0.5x ++ 5100.213 CT 0.1x + 510 0.046 0.02x + 510 0.013* 0.01x +/− 510 0.005x − 510

The buffer of Table 4 has absorbance maximum at 510 nm which is close toFAM and SYBR fluorescence maxima. Absorbance would decrease qPCR signalswith these dyes significantly. Thus, the use of this mix in qPCR wouldnot be feasible.

TABLE 5 Product Dilution Visual color nm (λ_(max)) A_((1 mm)) A_((1 cm))Green GoTaq 5x +++ 419 Promega 1x ++ 419 1.183 0.5x ++ 419 0.527 GT 0.1x++ 419 0.130 0.02x + 419 0.029* 0.01x + 419 0.014* 0.005x −

The mix of Table 5 has very strong absorbance at 419 nm. As theabsorbance peaks are not very sharp it also has significant absorbanceat 495 nm (0.17 with 1 mm light path), which is the range where FAM andSYBR dyes are excited. Absorbance would decrease qPCR signals with thesedyes. The absorbance spectrum (1× dilution) is shown in FIG. 5c .

TABLE 6 Visual Product Dilution color nm (λ_(max)) A_((1 mm)) A_((1 cm))Quick-Load mm 2x +++ 478 1.922* NEB 1x ++ 485 1.000 0.5x ++ 485 0.516 QL0.1x + 485 0.103 0.02x + 485 0.025 0.01x − 485 0.012* 0.005x 0.001x0.0005x

The mix of Table 6 has very strong absorbance at 485 nm. As theabsorbance peaks are not very sharp it also has significant absorbanceat 495 nm (0.982 with 1 mm light path), which is the range where FAM andSYBR dyes are excited. Absorbance would decrease qPCR signals with thesedyes significantly. The absorbance spectrum (1× dilution) is shown inFIG. 5d .

TABLE 7 Product Dilution Visual color nm (λ_(max)) A_((1 mm)) A_((1 cm))Coral Load 10x   +++ Qiagen 1x   ++ 475 0.407 0.5x ++ 475 0.202 CL0.1x + 475 0.043*  0.04x + 475 0.018  0.02x −

The mix of Table 7 has very strong absorbance at 475 nm. As theabsorbance peaks are not very sharp it also has significant absorbanceat 495 nm (0.393 with 1 mm light path), which is the range where FAM andSYBR dyes are excited. Absorbance would decrease qPCR signals with thesedyes significantly. The absorbance spectrum (1× dilution) is shown inFIG. 5 b.

Perceivable range is dependent on the wavelength but in general colorproviding a absorbance above 0.01-0.1 with 1 mm light path seems to bevisually distinguishable from the clear liquid. When absorbance israised to 0.1-0.2, the color appears very clear to the eye. However,sophisticated instruments are more sensitive and thus dyes providingabsorbance above 0.001 could be used when e.g. a spectrophotometer isused for color measurement.

Use of instrument for checking the reaction setup volume by colordetection enables more diluted colors to be used for that purposeminimizing possible negative effects that the colors might have. Forexample in qPCR the range of dyes that could be used withoutsignificantly affecting fluorescence detection would be increased.

The embodiments and examples above and the attached drawings are forillustrative purposes. The scope of the invention should be evaluated onthe basis of the following claims taking equivalents into account.

Applicants incorporate by reference the material contained in theaccompanying computer readable Sequence Listing identified as SequenceListing 078623_13_ST25.txt, having a file creation date of Aug. 24, 201510:18 A.M. and file size of 1,033 bytes.

The invention claimed is:
 1. A solution set for a polymerase chainreaction (PCR) assay, the solution set comprising a first reagentsolution comprising a first colorant having a first colordistinguishable in the visible spectrum, and a sample solutioncontaining a template to be amplified, and a second reagent solutioncomprising a second colorant having a second color distinguishable inthe visible spectrum, the second color different from the first color,and at least one substance selected from the group consisting of apolymerase, a primer, a plurality of dNTPs, a fluorescent qPCR dye, anda probe, at least the first and second reagent solutions on mixingforming a mixed solution, the mixed solution having, due to the firstand second colorants, a third color different from the first and secondcolors and distinguishable in the visible spectrum.
 2. The solution setof claim 1 where the first reagent solution is a dilution buffer.
 3. Thesolution set of claim 1 where the second reagent solution is a PCRmaster mix or a qPCR master mix.
 4. The solution set of claim 1 where atleast one of the first or second colorants is a dye.
 5. The solution setof claim 1 where the second reagent solution is a PCR master mix or aqPCR master mix and the first reagent solution is a dilution buffer. 6.The solution set of claim 5 where the first color is blue, the secondcolor is yellow, and the mixed solution color is green.
 7. A solutionset for a polymerase chain reaction (PCR) assay, the solution setcomprising a first reagent solution comprising a first colorant having afirst color distinguishable in the visible spectrum, and a PCR mastermix or a qPCR master mix, and a second reagent solution comprising asecond colorant having a second color distinguishable in the visiblespectrum, the second color different from the first color, and a samplesolution containing a template to be amplified, the first and secondreagent solutions on mixing forming a mixed solution having, due to thefirst and second colorants, a third color distinguishable in the visiblespectrum and different from the first and second colors.
 8. The solutionset of claim 7 where the sample solution is a dilution buffer.
 9. A kitcomprising the solution set of claim 1 and instructions for performingthe PCR assay.
 10. A kit comprising the solution set of claim 7 andinstructions for performing the PCR assay.
 11. The solution set of claim1 where the at least one the first or second colorants is selected fromthe group consisting of Quinoline yellow, Xylene cyanol, Brilliant Blue,Patent Blue, Indigocarmine, Acid Red 1, m-Cresol Purple, Cresol Red,Neutral Red, Bromocresol Green, Acid Violet 5, Bromo phenol blue, andOrange G.
 12. The solution set of claim 1 where the concentration of thecolorant in each of the first reagent solution and the second reagentsolution results in an absorbance of 0.001-0.5 at a maximum absorptionwavelength of the reagent solution with a 1 mm light path when dilutedto a desired PCR processing concentration.
 13. The solution set of claim12 where the absorbance is 0.01-0.5.
 14. The solution set of claim 12where the absorbance is 0.03-0.15.
 15. The solution set of claim 1 whereat least one of the first or second reagent solutions is a concentrate.16. The solution set of claim 1 where the first reagent solution and thesecond reagent solution form a transparent or translucent mixed solutionsuitable for qPCR.
 17. The solution set of claim 1 where the mixedsolution comprises a fluorescent agent, and where an absorbance peak ofany colorant does not overlap with an emission or excitation wavelengthof the fluorescent agent.
 18. The solution set of claim 1 where themixed solution comprises a fluorescent agent and a total absorbance ofthe mixed solution at the emission or excitation wavelength of thefluorescent agent is less than 0.05 with a 1 mm light path.
 19. Thesolution set of claim 18 where the total absorbance of the mixedsolution at the emission or excitation wavelength of the fluorescentagent is less than 0.03.
 20. The solution set of claim 18 where thetotal absorbance of the mixed solution at the emission or excitationwavelength of the fluorescent agent is less than 0.01.
 21. The solutionset of claim 1 where the fluorescent qPCR dye comprises a passivereference dye.
 22. The solution set of claim 21 where the passivereference dye is ROX.
 23. The solution set of claim 7 where the PCRmaster mix or the qPCR master mix further comprises a passive referencedye.
 24. The solution set of claim 23 where the passive reference dye isROX.